1. Field of the Invention
The present invention relates to a novel, simple and accurate method for studying interaction of a native ligand and its receptor using a fluorescently labeled, indicator ligand and a porous, optically transparent matrix that can be permeated by the fluorescently labeled ligand but is partially or totally impermeable to the labeled ligand when it is bound to the receptor. Fluorescence emitted from within the porous matrix is then useful as an indicator of the interaction between the native ligand and the receptor.
State 2. of the Art
Interaction of a ligand and a receptor in solution has been assayed or studied in the past by complicated techniques using differently labeled components. One such method is to use a radioactively labeled ligand or receptor. Another method is to utilize a fluorescent dye that is attached to the ligand or receptor. Generally, however, it has been necessary to isolate the reactants or products by complicated procedures. For example, special procedures have been necessary to separate bound from unbound ligands.
In U.S. Pat. No. 4,816,419 a method for fluorescence ligand binding assays is disclosed wherein certain surfactants that form micelles in solution were found to be useful in effecting differential fluorescence between bound and unbound ligands. The micelles sequester bound from unbound labeled ligands. Capture of labeled ligands occurs preferentially for free labeled ligands. When the labeled ligands are bound to a receptor, capture by the micelles is severely inhibited. The fluorescent emission is different when the labeled ligands are captured by the micelles than when the bound labeled ligands are not captured.
The method using the micelles of U.S. Pat. No. 4,816,419 is severely restricted in the breadth of ligands and receptors that can be analyzed or studied. The method employing the micelles is chemical in nature, thereby being restricted to the study of ligands and receptors bound to the ligands that interact in specific ways with the micelles. The ligands must be bound to or combine with the micelles, and the ligands bound to the receptor must not be attracted to or combine with the micelles.
3. Objectives
A principal objective of the invention is to provide a novel, simple, accurate, fast, economical method of studying and assaying the interaction of two molecules, such as a ligand and its receptor, in solution utilizing the measurement of fluorescence within an optically transparent matrix that is differentially permeable to the molecules depending upon the size of the molecules.
A particular objective of the present invention is to provide such a method of studying the interaction of two molecules in solution wherein a fluorescently labeled molecule is used that can permeate a porous, optically transparent matrix, with the reaction product of the labeled molecule and the other molecule being partially or totally excluded from the matrix, wherein the matrix is then viewed, such as through a fluorescent microscope, to determine fluorescence emitted from within the matrix.
Another objective of the present invention is to provide such a method of studying the interaction of two molecules in solution that does not rely upon chemical dependency of either of the molecules with the means being used to differentiate between the molecules, but rather utilizes the physical size of the molecules such that one of the molecules permeates a porous, optically transparent matrix but the other molecule is partially or totally excluded from the matrix.